Nucleosomes are "transparent" to transcription by RNA polymerase II (Pol II) in vivo, but not in vitro. The nature of the strong in vitro barrier to Pol II is unknown. Nucleosomes survive transcription in vivo, but passage of Pol II generates an "open" chromatin state where a high-order nucleosome structure is disturbed. During transcription, the individual nucleosomes change their position on DNA, some histones dissociate from the histone octamer, and the histone tails become chemically modified. The relationship between all these alterations and the strength of the nucleosomal barrier is mostly circumstantial. This is due to the lack of an in vitro system where the biochemical details of the process can be analyzed. We accomplished this goal by designing the "minimal" in vitro system where core enzymes of the yeast Pol II, Pol III, and E. coli RNA polymerase (RNAP) transcribe through a positioned mononucleosome.